Human papillomavirus (HPV) (FIG. 1) is a small DNA virus, and there are 100 or more genotypes. Fifteen genotypes (high-risk genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 73) cause cervical cancer. HPV16 is detected in 50 to 60% of cervical cancer. HPV18 is next frequently found in Europe and the United States, while HPV52 and HPV58 are next frequently found in Japan. Although mass examination was carried out for earlier diagnosis in Japan, there are still onset thereof in 15,000 patients and death of 2,500 patients every year.
The HPV capsid has a regular icosahedral skeleton consisting of 72 L1 protein pentamers (capsomeres) and 12 L2 protein molecules bound thereto. The both terminals of the L2 protein are located in the capsid, but part of the N-terminal region is located on the surface of the capsid (L2 surface region) (FIG. 2). Expression of the L1 protein in a great amount by recombinant DNA technique gives virus-like particles (VLPs). Inoculation of the VLP or the L2 protein of bovine or cottontail rabbit papillomavirus makes the inoculated animals resistant to viral challenge. FIG. 2 is a schematic cross-sectional view illustrating the HPV and the VLP.
There is currently no cultured cell line allowing proliferation of the HPV. Pseudoviruses are prepared for monitoring HPV infection. Introduction of a secretory alkali phosphatase (SEAP)-expressing plasmid having the replication origin of SV40, an L1 protein-expressing plasmid, and an L2 protein-expressing plasmid into SV40T antigen-expressing human 293 cell leads to incorporation of the replicated SEAP-expressing plasmid into the L1/L2 capsid, giving an infectious pseudovirus (FIG. 3). The activity of neutralization antibodies is determined by measuring the activity of inhibiting the pseudoviral infection (Nonpatent Document 1).
Each of the antisera obtained by inoculation of the VLP of HPV into animals has a type-specific neutralization activity. Merck developed a vaccine in combination of the VLPs of HPV16 and HPV18 and the VLPs of HPV6 and HPV11, which are possible causes of condyloma acuminatum (benign), while GlaxoSmithKline developed a vaccine in combination of the VLPs of HPV16 and HPV18 (Nonpatent Documents 2 and 3).
These vaccines were shown in large-scale clinical tests to have type-specific infection-preventing action, and the vaccine of Merck was approved in 2006 by FDA and Commission of the European Communities and sold in the U.S and EC countries.
As described above, immunization of the HPV L1 capsid into animals leads to induction of extremely type-specific immune response. Preliminary results in a clinical test by using the HPV16 L1 capsid vaccine showed that the vaccine was effective in preventing HPV16 infection, but almost not effective in preventing other HPV genotypes. Accordingly for prevention of onset of cervical cancer with vaccines, there is a need for development of a vaccine antigen that is effective at least to all high-risk HPVs.
The inventors had earlier developed a vaccine antigen, by using a common neutralization epitope to high-risk HPVs that is present in the amino acid 108-120 region of HPV16 L2 protein. However, the epitope has an amino acid sequence having a homology of about 60 to 75% with high-risk L2 proteins, and the antibody induced thereby bound to multiple high-risk HPVs but was lower in binding efficiency than to HPV 16. Thus, there has been a demand for an antigen having higher type-commonality.
The inventors had found that it was possible to prepare a vaccine antigen capable of inducing a more potent type-common neutralizing antibody by producing a chimeric protein having the HPV16 L1 protein and the amino acid 64-81 region of HPV16 L2 protein inserted thereto and that the vaccine antigen was an antigen having higher type-commonality that was compatible at least with all high-risk HPVs, and filed an patent application (WO2007/018049, Patent Document 1) earlier. Further as described above, in view of the fact that the particles formed of the chimeric protein composed of the HPV 16 L1 protein and an amino acid 108-120 region of HPV16 L2 protein inserted in the HPV16 L1 protein, have strong immunogenicity and potential to induce neutralizing antibody, which is in common to high-risk HPVs, a chimeric protein consisting of the chimeric protein having the additional amino acid 64-81 region inserted by an amino acid 109-117 region of HPV16 L2 protein was provided in Patent Document 1.    Nonpatent Document 1: Pastrana, D.V., Buck, C.B., Pang, Y.Y., Thompson, C.D., Castle, P.E., FitzGerald, D.C., Kruger, Kjaer, S., Lowy, D.R., Schiller, J.T., 2004. Reactivity of human sera in a sensitive, high throughput pseudovirus-based papillomavirus neutralization assay for HPV 16and HPV 18. Virology. 321: 205-216.    Nonpatent Document 2: Villa, L.L., et al.: Prophylactic quadrivalent human papillomavirus types 6, 11, 16, and 18) L1 virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre phase II efficacy trial. Lancet Oncology 6, 271-278, 2005    Nonpatent Document 3: Harper, D.M., et al.: Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus type 16 and 18: follow up from a randomized control trial. Lancet 367 (9518), 1247-1255.    Patent Document 1: W02007/018049All descriptions in the Nonpatent Documents 1 to 3and Patent Document 1 above are incorporated herein by reference.
HPV vaccines currently commercially available are effective only to HPV16 and HPV18 among 15 high-risk HPVs. Each VLP induces a type-specific neutralizing antibody, and thus, a cocktail of 15 kinds of VLPs are needed for induction of antibodies to all high-risk HPVs, making it difficult to prepare a practical vaccine antigen. Therefore, there is a demand for development of a vaccine antigen that induces a cross-reactive neutralizing antibody.
The antigen described in Patent Document 1 is an antigen compatible at least with all high-risk HPVs that have higher type-commonality. However, it had the following problem. The infectious pseudoviruses used in the study of Patent Document 1 were only HPV16 and HPV18, and the antigen described in Patent Document 1 showed favorable neutralizing activity to these infectious pseudoviruses. However, in the subsequent neutralizing experiment by using HPV31, HPV52 and HPV58 infectious pseudoviruses newly developed by the inventors, the antigen described in Patent Document 1 was lower in neutralizing activity to HPV31, HPV52, and HPV58.
Thus, an object of the present invention is to provide a vaccine antigen capable of inducing a cross-reactive neutralizing antibody to high-risk group human papillomaviruses.